Reading of fluorescent arrays

ABSTRACT

Reading of fluorescent arrays ( 103 ) in clinical settings is made possible by a reader ( 110 ) constructed to employ dark field illumination of the array, and mapping an image of the array onto a solid state sensor array ( 146 ) with image dimensions (D;) of the same order magnitude as the dimensions (D( ) of the fluorescent array, preferably with reduction of image. High intensity illumination is employed, non uniformities of which being compensated by normalization employing intensity calibration features ( 164 ) in the array itself, that are sensed during imaging of the array. Preferably high intensity light emitting diodes ( 122, 132, 402, 404 ), such as used in traffic lights, are employed for excitation of the array, preferably the excitation being introduced to the array via a solid internally reflecting homogenizer ( 130 ). Intermediate depth of field collection and imaging optics enable substantial collection of light, with NA in the range of 0.30 to 0.60, preferably in the range of 0.4 to 0.55. The resultant relatively large depth of field is in some advantageous cases compensated by absorbing light that tends to travel beyond the spots being imaged and would otherwise create noise fluorescence, the absorption produced e.g., by an opaque metal oxide coating ( 304 ) that is interposed between a substrate ( 302 ), preferably an ultra-thin substrate, on which the array lies, and the much thicker glass or other rigid support ( 306 ). For clinical purposes the arrays comprise fewer than 1000 spots, as is appropriate for protein, one example being an array of fewer than 500 spots. Relatively large spot sizes are employed, i.e. of the order of at least 80 or 100 micron diameter spots or preferably larger, 150 or 300 micron spots. Resolution of such spots to at least 50 pixels on the solid state detector array enables suitable binning and other manipulations leading to highly accurate results. Novel methods of assays and diagnosis such as cancer diagnosis employ the reader in detecting a set of markers related to the disease, for instance ovarian cancer.

TECHNICAL FIELD

The field to which this disclosure relates is clinical micro-arraytechnology, for instance clinical research and clinical diagnosis.

BACKGROUND

Micro-array technology has developed over the past decade and more. Itis employed in the investigation of biological molecules, in particular,nucleic acid and amino acid materials. Effective use has been made ofthe technology in understanding the genome and in drug discovery. It hasbeen predicted that the technology would ultimately develop to enablepractical use in the clinic, e.g. for clinical investigations andclinical diagnosis, but that prospect has seemed far off. Among reasonsfor this being only a long-term hope has been the very high cost of therequired equipment, the time involved in carrying out assays, and thehigh level of experience and skill required.

As is well known, micro-arrays are used by creating a field of featuresor spots of different analytes that are tagged or marked if certaincomponents are present. While marking has often been by radioisotopetags, fluorescent tags have come into wide usage for a number of reasonsincluding the ease by which the materials can be handled and out ofsafety considerations. Typically it is desired to represent an assay bya complete image of an array, or small set of related arrays.

The reader of fluorescent micro-arrays is key to the use of the arrays.The reader records presence and degree of fluorescence at each of theprecisely located features in the array in response to exposure tophotoexcitation. After consideration of numerous arrangements for readerdesign, a few successful technologies have found acceptance. Theseuniversally have required precise and costly mechanical movements, aswell as extensive optics or software. In one case, a rapidly oscillatingscanner arm moves a tiny lens for reading one pixel at a time in onecoordinate, in a confocal configuration, while the image of the array inthe other coordinate is developed by precise, gradual advance using amicroscope stage. (The stage is a device that creates precise movementsof micron or sub micron accuracy and is costly to manufacture and alongwith the other components.) With this approach, software is used toassemble the image from the vast array of gathered pixels. Anothertechnique has been to image highly magnified views of portions of theoverall array, by use of precision stage movement between the taking ofeach of the series of magnified images of the small portions of thearray, and then electronically merging or “stitching” the small fieldimage frames together to electronically produce an image of the completearray. Prior proposals or speculation for employing a solid state arrayof sensors to image an entire array at one taking have not resulted inpractical solution of the entire set of problems, i.e., simultaneouslyachieving high accuracy and high speed of operation at reasonable cost.As time has passed, and volume of production of readers has increased,the cost of those imaging systems that are successful, by elegantdesign, has been reduced from hundreds of thousands of today's U.S.dollars, in some instances to cost somewhat under one hundred thousanddollars. However, the prospect has seemed far off when volume productionwould enable the price for the readers to approach the cost that mightmake clinical usage attractive, e.g. a price of the order of twenty fivethousand U.S. dollars or less.

SUMMARY

In general, a reader capable of practical clinical use, i.e. in clinicalresearch, clinical diagnosis and monitoring, is found to be possiblewith a certain combination while observing certain constraints, and itcan be implemented with generally available components found in otherfields. Embodiments can satisfy the crucial low cost need for a clinicalreader, along with need for reasonably high speed and ease of operation,and while achieving the high level of accuracy required for medical use.

It is realized that the basic reader geometry should be based on darkfield illumination, e.g. light reaching the two-dimensional array at anacute angle of 20 to 50 degrees, which should be mated withtwo-dimensional imaging on a solid-state detector array, such as that ofa CCD sensor or CMOS array, with mapping on that array being of a scaleof the same order of magnitude as the array of biological features.Imaging is performed at normal angle to the plane of the biologicalarray, and is achieved with an optical collection and imaging systemhaving an intermediate range numerical aperture (NA), preferably betweenNA=0.3 to 0.6, and presently, preferably within the range of NA=0.4 to0.5.5. Embodiments within these constraints are capable of imaging anentire array in a single frame, without movement or stitching ofcomponents of the array. It is realized that cooperation of the featuresin the instrument, preferably with further novel enhancements to bedescribed, can make up for the inherent limitations of such anarrangement, i.e. its relatively large depth of field, and, when usingpreferred relatively inexpensive lighting, such as by high intensitydiodes, its non-uniform illumination. The resulting apparatus, becauseof its simplicity and lack of precise moving parts or expensive optics,can be made available to clinics at a cost that makes the system andtechnique practical. In such manner, practical, high-speed clinicalimaging is made possible even now, and from this, great benefits tomedicine and patient care can be obtained.

According to one aspect of the invention, an array reader is providedthat is suitable for clinical purposes for reading a two-dimensionalarray of features on a planar substrate, in which the features carryphoto-responsive markers, the markers capable of emitting light uponexcitation, the array reader comprising an illumination system forsimultaneously exciting multiple photo-responsive markers distributed ina two-dimensional array over the substrate, and an image collection andrecording system having a field of view for emissions from the markerson the substrate, wherein the illumination system comprises a lightsource arranged to flood the two-dimensional array with light at anexcitation wavelength, along an illumination path disposed at an angle θbetween about 20 and 50° to the plane of the substrate, the imagecollection and recording system having an image-acquiring axissubstantially normal to the plane of the substrate carrying the array,employing a two-dimensional sensor comprising a solid-state array ofphotosensitive elements, e.g. a charge-coupled device (CCD) or a CMOSarray, and the image collection and recording system constructed andarranged to apply an image of the array of markers upon the solid-statearray of size of the same order of magnitude as the size of the array,e.g. within a range of magnification of up to about 25% or reductiondown to about 75%, the image collection and recording system having anintermediate numerical aperture NA, to enable recording the image offluorescence from the excited two-dimensional array with clinicalaccuracy and without translation of the array.

Preferred embodiments of this aspect of the invention have one or moreof the following technical features.

The array reader image collection and recording system has its nearestcomponent spaced at least 5 mm, preferably at least 10 millimeter, fromthe substrate or its support, the component constructed and arranged toprovide space below the component for the illumination path to thetwo-dimensional array on the substrate.

The image collection and recording system has an effective aperturebetween NA=0.3 and NA=0.60, preferably the value of NA being betweenabout 4.0 and 5.5.

The image collection and recording system has a field of view on thesubstrate of areas between about 50 mm² and 300 mm².

The illumination system comprises one or more light-emitting diodes.

The illumination system, and especially the diode-based system, isconstructed and arranged to provide excitation illumination over thetwo-dimensional array on the substrate of a power density greater than30 mW/cm² and preferably the image collection and recording systemincludes a timer cooperatively related to the illumination system toprovide exposure sufficient to produce a fluence of excitation radiationat the substrate greater than about 15 mJ/cm² across the two-dimensionalarray.

The array reader has a field of view of diameter of the order of 10 mmor more.

Each feature of the array of interest is imaged onto a minimum of 50pixel elements of the solid state array, for example upon CCD or CMOSelements. (In the preferred case shown here the pixels (i.e. sensorelements) of the solid state array are selected to be of 9 microndimension, albeit, if larger field were to be imaged, using the samearrangement, pixels down to about 4.5 micron may be selected, and morereduction of image may be employed).

The array reader is constructed and arranged to deliver to the solidstate sensor an image of the field of view that is not magnified,preferably the reader being constructed and arranged to deliver to thesolid state sensor array an image of the field of view reduced betweenabout 30% and 50%.

The array reader is constructed to image spots each of diameter at leastabout 80 micron, preferably at least 100 micron diameter.

The array reader is constructed and arranged to produce, during a singleimaging interval, an image of an array of at least 100 spots each of 300micron diameter, or of at least 400 spots each of 150 micron diameter.

The array reader is combined with a carrier for the array comprising asubstrate layer carried by a support body, the image collection andrecording system residing on the same side of the substrate as does thearray of features such that the path of illumination reaches the arraybefore reaching the support body, the carrier constructed to absorbexcitation radiation penetrating beyond the layer, preferably thesupport body being transparent, e.g. glass, and between the substratelayer and the transparent body resides a substantially opaque adherentlayer capable of substantially blocking excitation radiation tending toenter the transparent body, preferably the substantially opaque layercomprising a layer of metal oxide.

The array reader is combined with a carrier for the array in the form ofa transparent layer carried by a transparent body, the image collectionand recording system lying beyond the transparent body on the same sideof the array as the transparent body.

The array reader is combined with a carrier for the array that comprisesan ultra-thin substrate layer on a support body, i.e. the substratehaving a thickness less than 5 micron, preferably less than about 3micron.

The array reader is combined with a carrier in which the array isdisposed on a substrate comprising a clear layer of nitrocellulose orpolystyrene.

The array reader is combined with a carrier in which the substrate is anitrocellulose membrane that is porous at least in its outer region.

The array reader is combined with a substrate carrying excitation energyreference features distributed across the two-dimensional array offeatures, the image collection and recording system including anormalizing arrangement for normalizing data detected in the vicinity ofrespective reference features based on the quantity of detected emissionfrom the respective reference features.

The array reader has an illumination system which comprises at least twodifferent light source sub-systems respectively of substantiallydifferent wavelengths, each associated with a respective optical systemdelivering light along a path, the paths of the sub-systems to thesubstrate lying along respectively different axes, the axes being spacedapart about the substrate, in certain preferred embodiments there beingtwo different light source subsystems the paths of which are disposed ondiametrically opposite positions about the substrate.

The array reader has an illuminating system which includes light sourcesselected respectively to excite Cy3 and Cy5, and the image collectionand recording system includes changeable band-pass filters suitable topermit passage of emissions respectively from Cy3 and Cy5 or a singleband-pass filter is provided suitable to permit passage of multipleband-pass emissions such as both the band-pass emission of Cy3 and ofCy5.

The image collection and recording system of the array reader isadjustable between at least two settings, the first and second settingsconstructed and arranged respectively to form a single image of an arrayformat of dimensions 6.5 mm×9.0 mm and of an array format comprising twoseparated sub-windows, each of dimensions 4.5 mm×4.5 mm disposed withina 4.5×13.5 mm rectangle.

The array reader illumination system includes a diode light source and ahomogenizer effective to reduce variation in flux density across thefield of illumination, in certain preferred embodiments the homogenizercomprising an elongated transparent, internally reflective rod, whichmay be straight or curved and may have round, square or rectangularcross section, and be twisted or untwisted.

The array reader has an image collection and recording systemconstructed and arranged to resolve the image on the solid state sensorarray at resolution no finer than about 10 micron, in certain preferredembodiments the resolution being between about 12 and 15 micron.

The array reader has an image collection and recording system whichincludes an interference filter, collection optics of the systempreceding the filter constructed to direct collected rays in parallel tothe filter, and imaging optics constructed to image parallel raysleaving the filter upon the solid state sensor.

The array reader is constructed to be used with an array support thatholds more than one array, and wherein the reader is constructed andarranged to read and process each array as an independent array.

The invention also includes a method of conducting an assay comprisingpreparing a two-dimensional spotted array of amino or nucleic acidfeatures on a substrate, preferably by spotting liquid samples thereon,in which features in the array carry fluorescent markers and employingthe reader of any of the foregoing descriptions to read the array.

Preferred embodiments of this aspect of the invention have one or moreof the following technical features.

The assay is a diagnostic immuno assay based on protein derived fromblood, in certain embodiments preferably the immunoassay is of anantibody capture configuration, for instance adapted, by immobilizedantibodies to detect or monitor for malignant cancer, e.g. to detectovarian cancer for initial diagnosis or to monitor patients at risk forrelapse.

The substrate is disposed within a sealed disposable bio-cassette andimaging is performed through a transparent window visually accessing thesubstrate, or a transparent body forming a side of the bio-cassettecarries the substrate, the substrate being transparent and the arraybeing accessed visually by the array reader through the transparent bodyand through the substrate.

For any of the array reader embodiments described or for any of theforegoing methods, for reading an array on a substrate, in certainpreferred embodiments the array includes intensity calibration markersof fluorescing character generally proportional in emission intensity toexcitation level over the range of operable illumination intensities,and the system or method includes forming an image of the arrayemploying the array reader, and normalizing recorded array data based onquantitative data acquired from nearby intensity calibration markers.

Another aspect of the invention is a fluorescence reader-baseddiagnostic method for a disease for which there is a set of knownprotein biomarkers in blood or other body constituent, comprising thesteps of (1) providing a two-dimensional array of different reagents ona substrate, the reagents respectively specific to bind members of a setof the biomarkers capable of diagnosing the disease, (2) exposing thearray to fluorophore-labeled blood or body-constituent extract of anindividual containing the biomarkers if present in the individual'sblood or body constituent, (3) while the array is stationary, excitingthe array by simultaneously illuminating the entire two-dimensionalarray by light at fluorophore-excitation wavelength, by employing darkfield illumination, (4) capturing a fluorescence image of the entiretwo-dimensional excited array on a single frame of an imager comprisinga solid state array, and (5) analyzing (e.g. by computer) thefluorescence image for the presence of the disease.

Preferred embodiments of this aspect of the invention have one or moreof the following features.

The method is performed in which the step of simultaneously illuminatingthe entire two-dimensional array is carried out by directing excitationradiation from a diode or set of diodes to produce illumination at awavelength selected to excite the fluorophore, at a power density of atleast 30 mW/cm².

The method is carried out in a way in which fluorescence intensityreference features are distributed through the array and the detectedradiation from the bio-markers is normalized by the reader based on theresponse of the references to the illumination.

The method is carried out in a way in which at least 50 pixels of thesolid-state sensor represent the image of a feature of the array.

The method is performed in which the biomarkers attach to antibodies.

The method is performed in a way in which the array is formed toimmobilize protein biomarkers selected to diagnose presence of ovariancancer.

Another aspect of the invention is a method of reading an array on asubstrate having features that include fluorophores, in which the arrayincludes intensity calibration features of fluorescing charactergenerally proportional in emission intensity to their illumination overthe range of operable illumination intensities, including, forming animage of the array employing an array reader, and normalizing recodedarray data based on quantitative data acquired during the reading of thearray from nearby intensity calibration features within the array.

Preferred embodiments of this aspect of the invention have one or moreof the following features.

The method is adapted to perform diagnosis for a disease for which thereis a set of known protein biomarkers in blood or other body constituent,comprising the steps of (1) providing a two-dimensional array ofdifferent reagents on a substrate, the reagents respectively specific tobind members of a set of the biomarkers capable of diagnosing thedisease, and including with the array the intensity calibration features(2) exposing the array to fluorophore-labeled blood or body-constituentextract of an individual containing the biomarkers if present in theindividual's blood or body constituent, (3) while the array isstationary, exciting the array by simultaneously illuminating the entiretwo-dimensional array by light at fluorophore-excitation wavelengthemploying dark field illumination, (4) capturing a fluorescence image ofthe entire two-dimensional excited array on a single frame of an imagercomprising a solid state array, (5) normalizing the recorded array databased on the calibration features in the array and (6) analyzing thefluorescence image for the presence of the disease.

The method is performed by illuminating the entire two-dimensional arrayfor forming the image by directing excitation radiation from a diode orset of diodes to produce illumination at a wavelength selected to excitethe fluorophore, at a power density of at least 30 mW/cm².

The method is performed under conditions in which at least 50 pixels ofa solid-state sensor represent the image of a feature of the array.

The method is employed to perform a diagnosis in which features of thearray include antibodies, in one important case the features of thearray are selected to diagnose the presence of ovarian cancer.

DESCRIPTION OF DRAWINGS

FIG. 1 is a diagrammatic view of an array reading system.

FIGS. 2A and 2B are diagrammatic views of illuminating devices for thereader.

FIG. 3 is a plot of relative intensity of illumination versus anglerelative to a central axis for a high intensity LED.

FIG. 4A depicts a spotting pin and reservoir suitable to form spots ofbiological material or intensity calibration spots, while FIG. 4Bdepicts an array in which the calibration spots are strategicallydistributed through the array of spots of biological material, FIG. 4Cbeing a magnified view, and FIG. 4D a plan view.

FIG. 5 depicts the mapping of a spot upon the array of solid statedetection elements of the sensor.

FIG. 6 is a diagrammatic representation, on highly magnified scale, of acarrier comprising a transparent rigid support body, bearing an opaquelayer, ultra-thin substrate and spots of the array on the substrate.Illumination from the same side as the array is shown.

FIG. 7A is a diagrammatic representation of a preferred clinical arrayreader, while FIG. 7B shows another clinical array reader.

FIG. 8 illustrates two array formats imageable by the array reader ofFIG. 7A.

FIG. 9 is a diagram representing the steps of an array-reading method.

FIG. 10 is a diagram representing the steps of a method for normalizingthe intensity level of pixels in the recorded image.

FIG. 11 is a diagram representing the steps of a fluorescencereader-based diagnostic method.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

Referring to FIG. 1, an array reading system 100 includes an arrayreader 110, a substrate 102 bearing a two-dimensional array of features103 (e.g. spots of bio-material such as amino or nucleic acid) some ofwhich, depending on the assay, carry fluorescent material, and acomputer 104 for processing images recorded with the array reader 110.The array reader 110 includes an illumination system 120 and an imagecollection and recording system 140.

During operation, the substrate 102 is positioned, with a positioner105, below the image collection and recording system 140, with adistance between them h that is large enough for light from theillumination system 120 to flood the two dimensions of the array 103,preferably h having the value of at least 5 mm and generally preferablyat least 10 mm. A preferred source of the illuminating light has anoutput between about 30 mW/cm² or more, and is preferably a lightemitting diode (LED) or array of such diodes. The features on thesubstrate contain material capable of emitting light within a narrowfluorescence spectrum upon excitation with light of selected wavelengthfrom the illumination system 120. Any available fluorescent dye may beemployed, presently Cy3 and Cy5 being common selections.

The image collection and recording system 140 collects the fluorescentlight and records a resulting image of the features or spots, theoptical system selected to produce a flat field of view. Theimage-acquiring axis 141 is substantially normal to the plane of thesubstrate 102. The illumination system 120 uses dark field illuminationsuch that light from the illumination system 120 is directed along apath that has an angle θ between about 20° and 50° to the plane of thesubstrate 102 to prevent illumination light reflected from the substrate102 from entering the image collection and recording system 140.Fluorescent light emitted from the spots is collected by imaging optics142 with an intermediate numerical aperture, e.g. between NA=0.30 to0.60, and in presently preferred embodiments, in the range betweenNA=0.40 and 0.55. (all in air) to increase the field-of-view and theamount of light collected. The imaging optics 142 project an image 144of the array 103 on a two-dimensional array of solid state detectingelements comprising sensor 146. This solid state array is of dimensionsof the same order of magnitude as are the dimensions of the array ofbio-material spots. The imaging optics system 142 is designed to havesuch a large field-of-view that the entire array 103 is mapped onto thesensor 146. Generally the two arrays are relatively sized such that aspot of bio-material is resolved on at least 50 pixels,preferably-with-spot size of the order of 100 micron, of the order of100 pixels, or with spot size of 300 micron, of the order of 300 pixels.

With such construction, the physical size D_(i) of the image 144 isapproximately the same as the physical size D_(o) of the array 103,biased toward reduction, i.e. preferably not magnified more than about25% or reduced more than about 75%. Presently, with reasonably low-costavailable components, the array is not magnified, and preferably isreduced in the range between 30% and 50%. This provides the veryimportant feature of there being no requirement to translate the array103 by a precision stage relative to the reader 110 to acquire the imageor stitch together multiple images of small sections to form a singleimage of the array 103.

Referring to FIG. 2A, in a preferred embodiment, the illumination system120 includes a high intensity LED 122 for emitting light within anexcitation band (e.g., green) designed to excite a fluorescence spectrumof a material in a spot (e.g., Cy3). An excitation filter 124 is used tofurther limit the excitation wavelength band. The spatial distributionof the light is shaped with an optical system such as a pair of lenses126 and 128 for near uniform illumination of the two-dimensional array103 on the substrate 102. To provide higher intensity illumination, morethan one LED can be distributed about the array 103, also arranged toemit light along a path that has an angle θ between about 20° and 50° tothe plane of the substrate 102.

Referring to FIG. 2B, in another preferred embodiment, the illuminationsystem 120′ includes a high intensity LED 132 for emitting light withinan excitation band designed to excite a fluorescence spectrum of amaterial in a spot. An excitation filter 134 is used to further limitthe excitation wavelength band. A homogenizer 130 with suitable lensesintegral with its ends (not shown) reduces variation in flux densityacross the field of illumination onto the two-dimensional array 103situated on the substrate 102. It comprises a solid transparent rodsuitably designed or clad to have 100% internal reflection and of lengthrelative to diameter selected to produce the desired homogenizationeffect, to render the distribution of illumination more uniformfunction. The homogenizer 130 accepts light up to an acceptance angle ofapproximately 45°. The end of the homogenizer 130 is arranged to emitlight along a path that has an angle θ between about 20° and 50° to theplane of the substrate 102.

There are enhancements that can importantly be combined with the readerto raise performance of the reader to make it practical in variousimportant contexts.

Using at least one, and preferably a distribution of energy calibrationspots in each array, the detected intensity for spots of an array can benormalized against the detected value of radiation received fromnear-lying calibration spots during image processing, thus enablingtolerance of non-uniformity in radiation, as may occur in a low costlighting system. The technique may also be effective to compensate forimprecise location of the array under the reader. FIG. 3 shows anon-uniform illumination pattern generated by a typical high-intensityLED, plotted as relative intensity as a function of angle from thecenter axis. The intensity of fluorescent light recorded by the solidstate sensor array at a location in the image corresponding to acalibration spot is used to infer the local illumination intensity,which is used to normalize the signal level recorded at neighboringspots on the sensor array. Different calibrating spots typically local,respectively, to different sets of spots of unknown intensity are used.

The calibration spots are preferably placed on the substrate along withthe array of biological spots for accurate relative placement. FIG. 4Adepicts a well of a microwell plate containing a fluorescent calibrationcomposition in which a pin 161 is dipped to receive the composition forspotting a substrate 102 e.g. polyimide polymer (Kapton™) dissolved in avolatile solvent. FIG. 4B shows diagrammatically a spotted array 103 ofbiological spots 166 among which is a pattern of fluorescent calibrationspots 164 produced with the composition of FIG. 4A. FIG. 4C is amagnified view of a portion of a substrate 102 containing, in additionto biological spots 166, fluorescent intensity calibration spots 164.FIG. 4D is a diagrammatic plan view, on an enlarged scale, of an array103 of biological spots 166, showing a relative arrangement ofcalibration spots 164. This arrangement enables normalization ofintensity variations across the entire array 103. The same intensitycalibration spots can also be used as spatial fiducials for locating thearray or the overall outline of the array may be employed for locatingit to the control system.

Taking advantage of the large size of the spots, hence the reasonablesize of their image on the solid state array, despite no magnification,another enhancement is the binning of pixels in the solid state arrayimage, which helps to average out random noise. The as-supplied on-boardbinning capability of a conventional CCD imager may thus be employed toenhance the accuracy of the reading. FIG. 5 shows a section of a CCDnear a border of the image of a spot 202 corresponding enablingresolution of a feature in the array by approximately 291 pixels.

Other important enhancements involve provision of special features andcharacteristics of the substrate and its support which reduceauto-fluorescence capture (i.e., fluorescent light collected fromsources other than the fluorescent material in the features of thearray). FIG. 6 illustrates important aspects of a preferred substrate102. An ultra-thin layer 302 of material (e.g., thinner than about 5microns, preferably less than 3 microns) is comprised e.g. ofnitrocellulose or polystyrene. As a film it is transparent, though inother cases an ultra-thin porous nitrocellulose membrane may beemployed. The substrate supports the array 103 of features. The beingultra-thin, it limits the amount of fluorescence emitted by thesubstrate layer 302 itself.

An opaque layer 304, such as sputtered metal-oxide helps to preventillumination light 300 from penetrating into, and exciting fluorescencewithin, the rigid support 306 below (e.g. the substance of a glassmicroscope slide). This helps to counter potential auto-fluorescencecapture from the support layer 306 caused by the large depth of focusdue to not using a high numerical aperture optical system.

The array reader 110 enables imaging of an array of fluorescentlylabeled proteins, as well as other potential widespread uses, such asimaging proteins labeled with luminescent tags, and with otherbio-materials labeled with fluorescent or luminescent tags. The arrayreader 110 may be used to advantage with viruses, peptides, antibodies,receptors, and other proteins; with a wide range of other labeledbiological materials including plant, animal, human, fungal and bacteriacells; and with labeled chemicals as well. The array reader 110 isdesigned for rapid imaging of immunoassay arrays of the size relevant toclinicians, with typically fewer than 1000 spots.

The array reader 110 also enables performing immunoassays of multiplebiomarkers (e.g., for ovarian cancer) simultaneously. Diseases with aset of known protein biomarkers in blood or other body constituents, cantherefore be diagnosed more easily. After providing a two-dimensionalspotted array of reagents on a substrate, the reagent spots are exposedto fluorophore-labeled blood or other body-constituent extracted from anindividual suspected of having the disease. The resulting array of spotsare then read by the array reader 110.

Referring to FIG. 7A, in a presently preferred embodiment two lightingsub-systems are provided at diametrically opposite positions about thearray position, employing light sources originally designed for trafficlights. Array reader 400 includes a LumiLed high intensity LED 402(Luxeon green 535 nm, 5 watt LED, part # LXHL-LM5C available fromLumileds Lighting U.S., LLC, San Jose, Calif.) with a green peakwavelength for excitation of Cy3, and a second LumiLed high intensityLED 404 (Luxeon red-orange 617 μm, 1 watt LED, part # LXH-MH1B) with apeak wavelength for excitation of Cy5. Both LEDs have a low temperaturecoefficient, of about 0.04 nm/deg C., and a narrow band peak wavelengthtolerance, typically 8 nm for Cy5 and 30 nm for Cy3. These LEDs areavailable with a 10% to 20% conversion efficiency and a typicalspecification of 110 mW of continuous-wave output power that can bepeaked by 50% for a second at low duty cycle to yield about 150 mW,nearly all within the pass bands of a Cy3 excitation filter 406 (Chromafilter part # HQ 535/50, available from Chroma Technologies, Rockingham,Vt.) and the Cy5 excitation filter 408 (Chroma filter part # HQ 620/60).The f/1 cone (marked by lines 150 at 30° in the illumination patternshown in FIG. 3) includes 21.5% of the light, or 64.5 mW, (a largercapture is possible with the 45° acceptance angle of the homogenizer ofFIG. 2B). However, all the light in the f/1 cone does not transferthrough an f/1 lens because of Fresnel reflections at the higher angles.With proper filtering and vignetting it is safe to expect 50 mW (about33% transfer efficiency) in a round beam 10 mm in diameter with lessthan 20% spatial intensity for the red LED 404. Tilting the beam by 45°spreads the light over an ellipse enclosing the desired 6.5×9.5 mm²area, so the power density is about 45 mW/cm². In ½ sec, that yields afluence of 22.5 mJ/cm². For comparison, the excitation energy of alaser-confocal microscope is approximately 5 mW per 10 micron diameterspot for about 7.5 microsecond, yielding a fluence of 48 mJ/cm². Similarperformances are obtained for the green LED 402 with peak absorption forCy3. In addition to high power and cost efficiency, LEDS have a longlife, and allow straightforward implementation of multi-colorfluorescence. The green LED 402 uses a pair of Kohler lenses 412, andthe red-orange LED 404 uses a pair of Kohler lenses 414, so that bothLEDs deliver a nearly uniform beam over the 6.5×9.5 mm² field-of-view ofthe array 103. The LEDs are mounted on heat sinks available from theirsupplier.

Positioning of the substrate 102 relative to the viewing axis of thereader is performed by a positioner 105, e.g. a Geneva drive, withspatial resolution e.g. of 0.1 or 0.2 millimeter having a positionalaccuracy for instance of about 0.1-0.2 mm. The positioner 105 can beused to automatically shift from imaging one array to another, either onthe same or a different substrate, but of course is not of the precisionor cost of a microscope stage and plays no part in generating thecomponents of an image of the array. The same substrate can carry manyarrays without the need to precisely position the arrays relative to oneanother, and the positioner 105 acts to move one after another intoposition for imaging. Preferably, the substrate has alignment marks,“fiducials”, that aid in the positioning, for instance sets ofdistinctive marks that designate the corners of rectangular arrays.

The array 103 is imaged onto the CCD sensor 420 by a pair of commercialCCD lens assemblies 422, 422′ (Westech CCD lens assemblies #2105 and#2131, available from Westech Optical Corporation, Penfield, N.Y.), lens422 being used in an unusual way relative to the purpose of its originaldesign. A band-pass filter 424 (Chroma Technology part # 68030 for Cy5,Chroma part # 57030 for Cy3) located in between the two lensesselectively transmits only light within the excited fluorescencespectra. The image of one 6.5×9.5 mm² field-of-view (see 502, FIG. 8) isprojected onto the CCD sensor 420 reduced by a factor of 0.707, whereasthe image of two separated sub-arrays, lying in a rectangle 4.50×13.5 mmis reduced by a factor of 0.5.

The lenses are assembled to operate with a 0.42 NA on the object side(facing the array 103). The array 103 can be imaged onto the CCD sensor420 with the lens assemblies 422 assembled to operate with an NA aslarge as 0.52.

The CCD sensor 420 is cooled with a Peltier cooler 426 (as in theCCD-based camera from Santa Barbara Instrument Group, Inc., SantaBarbara, Calif., Model ST-7×ME) to reduce dark current noise. The cooler426 has the capability to cool the CCD sensor 420 to 50° C. belowambient if necessary. Despite the advantageous cooling, read-out noise,generated upon conversion of the stored charge in a pixel into avoltage, is a dominant source of noise and to the extent its effects arenot eliminated, read-out noise determines the minimum light intensitythat can be detected. As this noise is random and the fluorescent lightfrom the spots is not, most of its effects can be reduced by the “onboard binning,” dark field subtraction, time and frame integration,software analysis, and using a large number of pixels imaging each spot.

Referring to FIG. 7B, imaging in a dark field mode may also beaccomplished with direct illumination at angle θ as shown and CCD sensor24 positioned to view the array along axis A normal to the plane of thearray via collection optics 27, spaced a distance h from the substrate.In this case the substrate layer may be microporous partially orthroughout its depth or may be a solid film or a modified solid film,preferably in any of these cases being an ultra-thin coating or membraneof less than 5 micron thickness. As shown, light for direct illuminationenters along an illumination axis A′, at an acute angle θ to the planeof the array. Distance h must be selected to enable such directillumination, with angle θ ranging between about 200 and 50°, here shownat 45°. Light L originates from a source 112 a, 112 b or 112 c ofwavelength selected to excite the fluorophore tag of the array, passesvia dichroic mirrors 156 b, 156 c to mirror 116 located to the side thatdirects the illumination along axis A′ at angle θ, onto thefluorophore-tagged array of spots resident on the ultra-thin substrate20 or 20′. The array of spots may use a carrier that comprises anultra-thin substrate layer on a support body, or a carrier in the formof a transparent layer carried by a transparent body, the imagecollection and recording system lying beyond the transparent body on thesame side of the array as the transparent body. The fluorescentemissions are collected by lens 27, through a selected filter 28A, B orC, thence through lens 26 to CCD camera 24 under computer control 32. Asbefore, the background subtraction technique is used with this system.The differences between the excitation source and that of FIG. 7A, comeat a significant cost that is counter to the most cost-demandingsituations of the clinic, but the geometry and different capabilities ofthe lighting system of FIG. 7B can have advantages that enjoys the otherbenefits that have been described.

In an advantageous design, the immunoassay arrays are limited to 400microassay spots. The array format is then an important design issue.The size and number of pixels of the CCD's chip and the configurationsof commercial spotting arrayers are important constraints to be balancedagainst each other in the design of the reader and array. The size ofthe array must be matched to the CCD's parameters. On the other hand,spotter pin or tip configurations limit the choice of reservoirs forloading the spotting or printing head with source material, and alsolimit possible array configurations. Disposable microtiter plates witheither 96 or 384 wells are the typical reservoirs used. Theseconstraints are satisfied by the two formats presented schematically inFIG. 8.

Taking into consideration geometric tolerances, a first format 502field-of-view covers one 6.5×9.5 mm² array, and a second format 504field-of-view covers two 4.5×4.5 mm² arrays. Assuming 300-microndiameter spots, 500 microns on center, yielding a spot occupancy of 36%,each spot will be conjugated to about 291 pixels. Assuming 150-microndiameter spots, 333 microns on center, each spot will be conjugated toabout 91 pixels. The large number of pixels per spot permits the onboard 3×3 binning option available with the CCD sensor to increasesignal-to-noise ratio. The immediate background is subjected to the sameaveraging to yield a sensitive and reliable fluorescence signal level.Arrays can be formed with each of the formats using either of the twospot sizes. These arrays can be printed with all commercialarrayers/printers, such as the Affymetrix Pin and Ring Arrayer, startingwith either 96 or 384 microtiter plates as the source material loadingreservoir.

Other methods are useful to raise the signal-to-noise ratio to bestdefine the quality of the image. Longer integration time or the sum ofmultiple acquisitions of the stationary array are useful to avoid CCDsaturation. The signal-to-noise ratio improves as the square root of theratio of integration time or the number of frames. A 5 second read timeversus 0.5 seconds improves the signal-to-noise ratio by approximately3.16 times.

The signal-to-noise ratio can also be increased by increasing the numberof LEDs, e.g. to as many as 4 for each of the 2 wavelengths, to raisethe power level to 160 mW/cm2. Applied together, these options increasethe signal to noise ratio by as much as a factor of 13 by substantiallyraising the fluence to 1,120 mJ/cm². Photo-bleaching, a possibleconsequence, depending upon the dyes etc., may limit this approach inparticular circumstances.

Alternate embodiments can use other types of substrates. For example,the substrate can be a glass slide, or alternatively, a sealeddisposable bio-cassette where imaging is performed through a transparentwindow within the substrate.

Referring to FIG. 9, a method for multi-biomarker assay includes thestep 600 of providing a two-dimensional spotted array of amino ornucleic acid features on a substrate, where features throughout thearray carry photo-responsive sensitive markers. In the second step, 602,the illumination source of the array reader illuminates the array alongan illumination path at an angle θ between about 20 and 50° to the planeof the substrate. In the third step, 603, the image collection andrecording system then collects excited fluorescent light along animage-acquiring axis that is substantially normal to the substrate,followed by the step 604 of recording an image of the array ofbio-material spots on the solid array of a CCD sensor, followed by thestep 606 of normalizing the intensity level of pixels in the recordedimage using intensity calibration markers.

It is to be noted that this calibration occurs as an integrated actionin the imaging of each array. It is to be distinguished from pre-readingcalibration of the overall instrument, a normal but not totallyeffective procedure.

Referring to FIG. 10, a method for normalizing the intensity level ofpixels in the recorded image includes the step 1004 of determiningpixels that detect fluorescence from position calibration spots locatedat corners of an array 1002. The resulting position information is thenused to locate pixels that correspond to multiple “data sets” across thetwo-dimensional image. Each data set contains pixels corresponding tobiology spots, and pixels corresponding to an intensity calibrationspot. For each data set, including a “data set n,” the method includesthe step 1006 of detecting intensity recorded by pixels representing theintensity calibration spot n, the step 1008 of detecting intensityrecorded by pixels representing each biology spot in data set n, and thestep 1010 of normalizing the intensity data for each biology spot usingthe intensity of the intensity calibration spot. After intensity data isnormalized for each data set, in a final step 1012, the entire image isrepresented according to the normalized data for all of the data sets.

Referring to FIG. 11, a fluorescence reader-based diagnostic method, fora disease for which there is a set of known protein biomarkers in bloodor other body constituent, includes the step 1102 of providing atwo-dimensional array of different reagents on a substrate. The reagentsare respectively specific to bind members of a set of the biomarkerscapable of diagnosing the disease. The method then includes the secondstep 1104 of exposing the array to fluorophore-labeled blood orbody-constituent extract of an individual containing the biomarkers ifpresent in the individual's blood or body constituent. While the arrayis stationary, in a third step 1106, the reader excites the array bysimultaneously illuminating the entire two-dimensional array by light ata fluorophore excitation wavelength, employing dark field illumination.In a fourth step 1108, the reader then captures a fluorescence image ofthe entire two-dimensional excited array on a single frame of an imagercomprising a solid state array. The method then includes the step 1110of analyzing the fluorescence image for the presence of the disease.

In a preferred embodiment the assay is a diagnostic immunoassay based onprotein derived from blood, and can detect or monitor for malignantcancer, such as ovarian cancer, for initial diagnosis or to monitorpatients at risk for relapse. In the last decade, the search forbiomarkers that alone, or in combinations with Ca125, could improveprognostic testing for ovarian cancer yielded a number of candidates.However, in 1995, Berek and Bast reviewed data on 17 different markers(including CA125) and concluded that none was useful in the setting ofearly stage ovarian cancer (1). However, it has been shown that othertumor markers can complement CA125 and be useful in some circumstances(2).

Recently, genomic technologies have dramatically accelerated progress inthe search for prognostic ovarian cancer biomarkers. Studies usingdifferential DNA transcriptional profiling of ovarian cancer cell linesand those from ovarian epithelium collectively have identified hundredsof candidate biomarkers (e.g. 3, 4, 5). Some of these new candidateprotein biomarkers have been evaluated in exploratory trials. Candidatebiomarker proteins which have been studied include: HE4 (6), osteopontin(7), prostasin (4, 5) and mesothelin/megakaryocyte potentiating factor(8). Recent reports also suggest that members of the kallikrein serineprotease family, particularly kallikrein 10, may also serve as ovariancancer biomarkers in blood (9, 10, 11). Results from exploratory studiesare encouraging and suggest that these proteins either alone, or incombinations with other markers such as CA125, may be useful asprognostic indicators for ovarian cancer.

There is direct evidence that patterns of multiple biomarkers in bloodprovide a signal of early stage ovarian cancer. Proteomic spectragenerated by mass spectroscopy (SELDI-TOF) from sera of ovarian cancerpatients and normal individuals and analyzed by an iterative searchingalgorithm, identified a proteomic profile of five, of as yetunidentified proteins, that completely discriminated the sera of theovarian cancer patients (12). In a test of blinded samples, thediscriminatory pattern correctly identified 100% of the ovarian cancersamples including the 36% from early stage patients and showed aspecificity (false positive rate) of 95% (12).

If the positive predictive value of proteomic pattern technology issupported by population-based trials, these discriminating proteinsprovide excellent opportunities for developing highly sensitivediagnostic probes (13) which can, given the appropriate technologyplatforms, be ultimately exploited in routine tests for detecting earlystage ovarian cancer.

The references referred to are:

1. Berek, J S, R C Bast. 1995 Ovarian cancer screening. The use ofserial complementary tumor markers to improve sensitivity andspecificity for early detection. Cancer 76 2092-06.

2. Woolas, R P, D H Oran, A R Jeyarajah, R C Bast, J J Jacobs, 1999.Ovarian cancer identified through screening with serum markers but notby pelvic imaging. 1999. Int. J. Gynecol 9: 497-501.

3. Schummer, M, W V Ng, R E Baumgarner, PS Nelson, B Schummer, D WBednarski, L Hassell, B Y Baldwin and L. Hood. 1999. Comparativehybridization of an array of 21,500 ovarian cDNAs for the discovery ofgenes over-expressed in ovarian carcinoma Gene 238: 375-385.

4. Kwong-Kwok, W, R S Cheng, S C Mok. 2001. Identification ofdifferentially expressed genes from ovarian cancer cells by MICROMAX™cDNA microarray system. 2001. Biotechniques 30(3): 670-674.

5. Mok, S, J Chao, S Skates, K-K Wong, G K Yu, M G Muto, RS Berkowitz, DW Cramer. 2001. Prostasin, a potential Serum Marker for Ovarian Cancer:Identification Through Microarray Technology. JNCI 93 (19): 1458-1464.

6. Hellstrom, I, J. Raycaft, M Hayden-Ledbetter, J A Ledbetter, MSchummer, M McIntosh, C. Drescher, N Urban, K E Hellstrom. 2003. The HE4(WFDC2)-protein is a biomarker for ovarian carcinoma. Cancer Research63; 3695-3700.

7. Kim, J H, S J Skates, T Ude, K K Wong, J O Schorge, C M Feltmate, RSBerkowitz, D W Cramer, S C Mok. 2002. Osteopontin as a potentialdiagnostic biomarker for ovarian cancer. JAMA 287 (13):1671-9.

8. Scholler N, N Fu, Y Yang, Z Ye, G E Goodman, K E Hellstrom, IHellstrom. 1999. Soluble member(s) of the mesothelin/megakaryocytepotentiating factor family are detectable in sera from patients withovarian carcinoma. PNAS USA 96 (20): 11531-6. (found in the L. Hoodscreening study)

9. Shvartsman, H S, K H Lu, J Lillie, M T Deavers, S Clifford, J I Wolf,G B Mills, R C Bast, D M Gershenson, R Schmandt. 2003. Over expressionof kallikrien 10 in epithelial ovarian carcinomas. Gynecol Oncol 90:44-50.

10. Luo, L Y, D Katsaros, A Sorilas, S Fracchiolo, R. Riccinno, I ARigault de la Longris, D J C Howarth, E P Diamandis 2001. Prognosticvalue of human kallikrein 10 expression in epithelial ovarian carcinomaClin Can Res 7: 2317-2379.

11. Luo, L Y, D Katsaros, A Scorialas, S Fracchioli, R Bellino, M vanGramberen, H de Bruijn, A. Henrik, UH Stenman, M Massobrio, A G van derZee, I Vergote, EP Diamandis. 2003. The serumprognosis. Cancer Res 63:807-11.

12. Petricoin, E F, A M Ardekani, B A Hitt, P J Levine, V A Fusaro, M ASteinberg, G B Mills, C Simone, D A Fishman, E C Kohn, L A Liotta 2002.Use of proteomic patterns in serum to identify ovarian cancer. Lancet16: 359 (9306);572-77.

13. Wulfkuhle, J D, L A Liotta, E F Petricoin. 2003. Proteomicapplications for the early detection of cancer. Nature Reviews Cancer 3:267-75.

For further disclosure concerning the topics of (1) employing thecharacteristics of ultra-thin substrate layers in dark fieldillumination and imaging on a solid state array of sensors of size oforder of magnitude of the array of spots, in general and in particularof nitrocellulose and polystyrene, and their methods of manufacture anduse, (2) metal oxide and other absorbent layers beneath the substratethat absorb excitation light serving to enhance the operation or makepractical a clinical fluorescence reader and (3) formation andutilization of intensity calibration marks in micro-arrays for servingto enhance the operation of a fluorescence reader, in particular oneusing a high intensity light emitting diode or diode array forexcitation illumination, reference is made to a further PCT applicationbeing filed simultaneously herewith, which likewise claims priority fromU.S. Provisional Ser. No. 60/476,512, filed Jun. 6, 2003.

Other features and advantages of the invention will be understood fromthe foregoing and the claims and are within the spirit and scope of theinvention.

1. An array reader suitable for clinical purposes for reading atwo-dimensional array of features on a planar substrate, in which thefeatures carry photo-responsive markers, the markers capable of emittinglight upon excitation, the array reader comprising: an illuminationsystem for simultaneously exciting multiple photo-responsive markersdistributed in a two-dimensional array over the substrate, and an imagecollection and recording system having a field of view for emissionsfrom the features on the substrate, wherein the illumination systemcomprises a light source in the form of at least one light-emittingdiode arranged to flood the two-dimensional array with light at anexcitation wavelength, along an illumination path disposed at an angle(θ) between about 20° and 50° to the plane of the substrate, the imagecollection and recording system having an image-acquiring axissubstantially normal to the plane of the substrate carrying the array,employing a two-dimensional sensor comprising a solid-state array ofphotosensitive elements, and the image collection and recording systemconstructed and arranged to apply an image of the array of features uponthe solid-state array of size of the same order of magnitude as the sizeof the array, e.g. within a range of magnification of up to about 25% orreduction down to 75%, the image collection and recording system havingan intermediate numerical aperture NA to enable recording the image offluorescence from the excited two-dimensional array with clinicalaccuracy and without translation of the array.
 2. (canceled)
 3. Thearray reader of claim 1 in which the image collection and recordingsystem has an effective aperture between NA=0.3 and NA=0.60. 4-5.(canceled)
 6. The illumination system of claim 1 constructed andarranged to provide excitation illumination over the two-dimensionalarray on the substrate of a power density greater than 30 mW/cm². 7.(canceled)
 8. The array reader of claim 1 in which the field of view ofthe array reader has a diameter of the order of 10 mm or more. 9.(canceled)
 10. The array reader of claim 1 constructed and arranged todeliver to said solid state sensor array an image of the field of viewthat is not magnified.
 11. The array reader of claim 1 constructed andarranged to deliver to said solid state sensor array an image of thefield of view reduced between about 30% and 50%. 12-13. (canceled) 14.The array reader of claim 1 in combination with a carrier for the arraycomprising a substrate layer carried by a support body, said imagecollection and recording system residing on the same side of thesubstrate as does the array of features such that the path of saidillumination reaches said array before reaching the support body, saidcarrier constructed to absorb excitation radiation penetrating beyondsaid layer.
 15. The array reader of claim 14 in which said support bodyis transparent, and between said substrate layer and said transparentbody resides a substantially opaque adherent layer capable ofsubstantially blocking excitation radiation tending to enter thetransparent body.
 16. The array reader of claim 15 in which saidsubstantially opaque layer comprises a layer of metal oxide.
 17. Thearray reader of claim 1 in which said substrate is in the form of atransparent layer carried by a transparent body, the image collectionand recording system lying beyond the transparent body on the same sideof the array as the transparent body.
 18. The array reader of claim 1 incombination with a carrier for said array that comprises a substratelayer on a support body, the substrate having a thickness less thanabout 5 micron.
 19. The array reader of claim 1 in which said array isdisposed on a substrate comprising a clear layer of nitrocellulose. 20.(canceled)
 21. The array reader of claim 1 in combination with asubstrate carrying excitation energy reference features distributedacross said two-dimensional array of features, said image collection andrecording system including a normalizing arrangement for normalizingdata detected in the vicinity of respective reference features based onthe quantity of detected emission from the respective referencefeatures.
 22. The array reader of claim 1 in which said illuminationsystem comprises at least two different light source sub-systemsrespectively of substantially different wavelengths, each associatedwith a respective optical system delivering light along a path, thepaths of said sub-systems to said substrate lying along respectivelydifferent axes, the axes being spaced apart about said substrate. 23.(canceled)
 24. The array reader of claim 1 in which said illuminatingsystem includes light source diodes selected respectively to excite Cy3and Cy5, and said image collection and recording system includeschangeable band-pass filters suitable to permit passage of emissionsrespectively from Cy3 and Cy5 or a single band-pass filter is providedsuitable to permit multiple band-pass emissions of Cy3 and Cy5. 25.(canceled)
 26. The array reader of claim 1 in which said illuminationsystem includes a diode light source followed by a homogenizer effectiveto reduce variation in flux density across the field of illumination.27-38. (canceled)
 39. A fluorescence reader-based diagnostic method fora disease for which there is a set of known protein biomarkers in bloodor other body constituent, comprising the steps of (1) providing atwo-dimensional array of different reagents on a substrate, the reagentsrespectively specific to bind members of a set of said biomarkerscapable of diagnosing the disease, (2) exposing the array tofluorophore-labeled blood or body-constituent extract of an individualcontaining the biomarkers if present in the individual's blood or bodyconstituent, (3) while the array is stationary, exciting the array bysimultaneously illuminating the entire two-dimensional array by light atfluorophore-excitation wavelength employing dark field illumination, (4)capturing a fluorescence image of the entire two-dimensional excitedarray on a single frame of an imager comprising a solid state array, and(5) analyzing the fluorescence image for the presence of the disease.40. (canceled)
 41. The method of claim 39 in which fluorescenceintensity reference features are distributed through the array and thedetected radiation from said bio-markers is normalized by the readerbased on the response of said references to said illumination. 42-44.(canceled)
 45. A method of reading an array on a substrate havingfeatures that include fluorophores, in which the array includesintensity calibration features of fluorescing character generallyproportional in emission intensity to their illumination over the rangeof operable illumination intensities, including, forming an image of thearray employing an array reader, and normalizing recorded array dataduring the reading of the array from nearby intensity calibrationfeatures within the array. 46-50. (canceled)